how to express protein in e coli ?
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Rosetta H. Flagg
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Answer ( 1 )
To express protein in E. coli, researchers typically use a method called recombinant DNA technology. Here’s a simplified explanation:
Gene cloning: First, the gene encoding the protein of interest is isolated from its natural source or synthesized in the laboratory. This gene is then inserted into a plasmid, a small, circular DNA molecule that can replicate independently within bacterial cells like E. coli.
Transformation: The recombinant plasmid containing the gene of interest is introduced into E. coli cells through a process called transformation. This can be achieved using techniques such as heat shock or electroporation.
Selection and amplification: E. coli cells that have taken up the recombinant plasmid are selected for using antibiotic resistance markers present on the plasmid. These cells are then grown in culture to amplify the number of cells containing the recombinant plasmid.
Expression: Once a sufficient number of transformed E. coli cells are obtained, they are induced to express the protein of interest. This is often achieved by adding a chemical inducer to the culture media, which activates the promoter sequence controlling the expression of the gene on the recombinant plasmid.
Harvesting and purification: After expression, the E. coli cells are harvested, and the protein of interest is purified from the cellular components using techniques such as chromatography or affinity purification.
Analysis: The purified protein can then be analyzed using various biochemical and biophysical methods to confirm its identity, purity, and function.
By following these steps, researchers can effectively express proteins in E. coli for various purposes, including scientific research, biotechnology, and pharmaceutical development.